Crowding Stem Cells' Personal Space Directs Their Future

Johns Hopkins scientists report that restricting the shape and personal space of human stem cells from bone marrow is more important than any known molecular signal in determining the cell type they become.

Understanding the signals that tell stem cells what type of cell to become, and then harnessing those cues to get a single desired cell type, is key to any effort to use these or more primitive embryonic stem cells to regenerate or repair damaged tissue.

In the April issue of Developmental Cell, the Hopkins researchers report that mesenchymal (pronounced mez-EHN-kih-mal) stem cells forced to be spherical efficiently transform into precursors to fat cells, while those allowed to stretch and flatten move closer to becoming bone cells. These stem cells can naturally become fat cells, cartilage, bone cells, or smooth, cardiac or skeletal muscle.

"The types of cells that come from mesenchymal stem cells all have shapes specific to their functions, so we wondered whether the stem cells' shapes could actually direct their differentiation," says Christopher Chen, M.D., Ph.D., an assistant professor of biomedical engineering at Johns Hopkins. "The answer is that shape is critical to the stem cells' differentiation. It can actually induce molecular signals known to encourage fat cell or bone cell development and causes complete, uniform differentiation."

In the first week of laboratory studies, about 45 percent of stem cells forced to be round moved toward fat cell development, and 50 percent of spread-out cells got closer to being bone cells. By four weeks, all cells had followed the path dictated by their shape, Chen says, making shape the most powerful factor in whether human mesenchymal stem cells become fat or bone in the lab.

Ever since these stem cells were first isolated in the late 1990s, scientists have recognized that which cell type they become depends on the density at which they are grown in the lab. But while sparse growth was recommended to get bone cells, and congested growth was recommended to increase the amount of fat cells, no one knew why.

To really understand whether it was the cells' shape or some aspect of their neighbors that directed differentiation, M.D./Ph.D. candidate Rowena McBeath used a special technique, developed in Chen's lab, that restricts individual cells to small spaces without requiring cellular neighbors to do the crowding.

The technique, called micropatterning, uses technology that was initially developed for the semiconductor industry. Using a rubber-like material, stamps are created that each have a specific pattern of microscopic squares, each coated with a protein that attracts cells (fibronectin). The stamp is then used to transfer the pattern to a surface, resulting in "islands" to which cells stick. The researchers can precisely control the size of the islands, and consequently whether cells will form a ball or stretch out.

"With this tool we can restrict the ability of individual cells to spread, and we can do so thousands of cells at a time," says Chen.

McBeath's experiments showed that mesenchymal stem cells on the small islands balled up and, biologically speaking, moved closer to becoming fat cells, while those on large islands stretched out and got closer to becoming bone cells. In subsequent experiments, she proved that shape can't be overcome by known molecular signals traditionally used to encourage mesenchymal stem cells to differentiate into either fat or bone cell precursors.

"Stretching out pushes the stem cells toward becoming bone cell precursors, and no collection of fat-encouraging signals was able to subsequently overcome the early effect of shape," says McBeath, an M.D./Ph.D. candidate in the Cellular and Molecular Medicine graduate program.

McBeath also showed that a molecule called RhoA, known to be activated when cells spread out, can mimic the effect of shape on the stem cells' differentiation. Perpetually active RhoA caused the stem cells to move toward bone, while inactive RhoA pushed them toward becoming fat cells, even when exposed to factors known to encourage differentiation toward the opposite cell type.

"Remarkably, when the cells were simply grown in regular dishes in the lab, RhoA's activation or inactivation overrode signals usually used to direct their growth toward fat or bone," says McBeath. "But altering RhoA activity couldn't force a round cell to become a bone precursor, or a spread cell to become a fat cell on our micropatterns."

However, she discovered that activating the enzyme RhoA kinase or ROCK, which is turned on by RhoA, caused even balled cells to differentiate toward bone. On April 8, in recognition of her work, McBeath received the Nupur Dinesh Thekdi Research Award as part of Hopkins' 27th annual Young Investigators' Day.

Next, the researchers will work on figuring out exactly how shape dictates the stem cells' futures and what role ROCK and RhoA play in the process.

Authors on the report are McBeath, Chen, Dana Pirone, Celeste Nelson and Kiran Bhadriraju, all of Johns Hopkins. The research was funded by the National Institute of General Medical Sciences, the National Institutes of Health's Medical Scientist Training Program, the Ruth L. Kirschstein National Research Service Award, and The Whitaker Foundation.